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Natural Variation in Epigenetic Pathways Affects the
Specification of Female Gamete Precursors in Arabidopsis
OPEN
Daniel Rodríguez-Leal,a Gloria León-Martínez,b Ursula Abad-Vivero,a and Jean-Philippe Vielle-Calzadaa,1
a Grupo de Desarrollo Reproductivo y Apomixis, Laboratorio Nacional de Genómica para la Biodiversidad y Departamento de
Ingeniería Genética de Plantas, Cinvestav Irapuato CP36821 Guanajuato, Mexico
b Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional del Instituto Politécnico Nacional, Unidad Michoacán,
CP 59510 Jiquilpan, Mexico
In angiosperms, the transition to the female gametophytic phase relies on the specification of premeiotic gamete precursors
from sporophytic cells in the ovule. In Arabidopsis thaliana, a single diploid cell is specified as the premeiotic female gamete
precursor. Here, we show that ecotypes of Arabidopsis exhibit differences in megasporogenesis leading to phenotypes reminiscent
of defects in dominant mutations that epigenetically affect the specification of female gamete precursors. Intraspecific hybridization
and polyploidy exacerbate these defects, which segregate quantitatively in F2 populations derived from ecotypic hybrids,
suggesting that multiple loci control cell specification at the onset of female meiosis. This variation in cell differentiation is
influenced by the activity of ARGONAUTE9 (AGO9) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6), two genes involved in
epigenetic silencing that control the specification of female gamete precursors. The pattern of transcriptional regulation and
localization of AGO9 varies among ecotypes, and abnormal gamete precursors in ovules defective for RDR6 share identity with
ectopic gamete precursors found in selected ecotypes. Our results indicate that differences in the epigenetic control of cell
specification lead to natural phenotypic variation during megasporogenesis. We propose that this mechanism could be implicated
in the emergence and evolution of the reproductive alternatives that prevail in flowering plants.
INTRODUCTION
The life cycle of flowering plants alternates between a dominant,
diploid, sporophytic generation and a short-lived, haploid, gametophytic generation in specialized reproductive organs. Integration
between environmental signals and developmental programs that
control flowering initiates development of the female reproductive
lineage within the gynoecium. During early formation of the gynoecium in Arabidopsis thaliana, ovule primordia develop from the
placenta as finger-like protrusions by active cell divisions in the
subepidermal layer (Schneitz et al., 1995; Grossniklaus and Schneitz,
1998; Ferrándiz et al., 1999). A single cell is specified as the archeospore, which directly differentiates into the premeiotic gamete precursor known as the megaspore mother cell (MMC). The MMC
subsequently divides by meiosis to produce four haploid cells, one of
which is specified as the functional megaspore (FM), the first cell of
the female gametophytic phase. The FM develops by three rounds
of mitosis into a female gametophyte, containing three antipodal
cells, two synergids, the egg, and a binucleated central cell. After
double fertilization, the egg and the central cell will develop into the
embryo and the endosperm, respectively (Reiser and Fischer, 1993;
Grossniklaus and Schneitz, 1998; Drews and Koltunow, 2011).
This pattern of development characteristic of Arabidopsis and
the majority of flowering plants is known as the monosporic,
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Jean-Philippe VielleCalzada ([email protected]).
OPEN
Articles can be viewed online without a subscription.
www.plantcell.org/cgi/doi/10.1105/tpc.114.133009
Polygonum-type of female gametogenesis (Maheshwari, 1950;
Eames, 1961). Although the Polygonum-type prevails in most
angiosperms examined to date (Huang and Russell, 1992), there
are many examples of naturally occurring variations that affect cell
specification in the ovule during female meiosis or gametogenesis.
These variations often involve the emergence of several female
gamete precursors (Vandendries, 1909; Grossniklaus and Schneitz,
1998; Bachelier and Friedman, 2011), the incorporation of more
than one meiotically derived product to the female gametophyte
(Maheshwari, 1950; Madrid and Friedman, 2009), the formation of
nonreduced female gametophytes (Karpechenko, 1927; Bretagnolle
and Thompson, 1995; Ramsey and Schemske, 1998), or the formation of seeds through asexual reproduction by a process known
as apomixis that bypasses meiosis and fertilization (Grimanelli et al.,
2001; Koltunow and Grossniklaus, 2003). Although extensive comparative and morphological reports describing these alternatives are
available for many angiosperm taxa, the genetic basis and molecular
mechanisms that control this type of reproductive natural variation
remain unclear.
Several genes are known to be involved in the control of gamete
cell specification and meiosis, and they act either by restricting the
number of meiotic precursors or by enabling the progression
through the meiotic division (Sheridan et al., 1996; Ferrándiz et al.,
1999; Schiefthaler et al., 1999; Nonomura et al., 2003; Lieber et al.,
2011). In addition, epigenetic components mediated by the action of
small RNAs (sRNAs) have been described as important regulators
of cell specification and female gametogenesis in Arabidopsis
(Armenta-Medina et al., 2011; Rodríguez-Leal and Vielle-Calzada,
2012). sRNAs are 18- to 30-nucleotide RNA molecules that regulate
gene expression at the transcriptional and posttranscriptional level
by their association with members of the ARGONAUTE (AGO)
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protein family. Several classes of sRNAs have been defined, depending on their mechanisms of biogenesis and action (Ghildiyal
and Zamore, 2009). The function of AGO9 and other specific
members of the so-called RNA-directed DNA methylation or transacting small interfering RNA pathways are important for the correct
specification of female gamete precursors. Mutations in genes
such as RNA-DEPENDENT RNA POLYMERASE6 (RDR6), DICERLIKE3, and AGO9 exhibit increased frequency of abnormal gamete
precursors that often give rise to more than one female gametophyte developing in the Arabidopsis ovule (Olmedo-Monfil et al.,
2010). Additional roles for sRNAs and their interactors in female
meiosis and gametogenesis have been described, suggesting that
some epigenetic pathways are crucial for the establishment of the
gametophytic generation (Nonomura et al., 2007; García-Aguilar
et al., 2010; Olmedo-Monfil et al., 2010; Schmidt et al., 2011;
Tucker et al., 2012).
Here, we report a detailed analysis of the phenotypic effects of
natural variation in the control of gamete precursor specification in
the developing ovule of Arabidopsis. We show that in phylogenetically distant ecotypes, the mechanisms that specify gamete
precursors are naturally variable, and they often lead to the differentiation of supernumerary cells as premeiotic precursors. Moreover, the frequency at which these ectopic cell configurations occur
is increased in F1 hybrids of specific ecotypes, and the prevalence
of ectopic cells at late stages of megasporogenesis is increased in
tetraploid individuals. We also show that the genetic introgression
of mutations affecting the function of AGO9 or RDR6 is buffered by
allelic interactions in the ovule of ecotypic F1 hybrids and that the
complete loss of AGO9 activity disrupts this mitigating effect. Finally, we demonstrate that the patterns of transcriptional regulation
and protein localization of AGO9 are variable between ecotypes
and that the abnormal gamete precursors found in mutants defective in RDR6 share a cellular identity with the ectopic cells naturally found in specific ecotypes. Our results link previously
characterized epigenetic pathways to mechanisms of natural variation that affect the specification of female gamete precursors in
Arabidopsis.
RESULTS
Selected Ecotypes of Arabidopsis Show Natural Variation in
Cell Differentiation during Megasporogenesis, Which Is
Influenced by Intraspecific Hybridization and Ploidy
Previous reports showed that in most cases of megasporogenesis
in Arabidopsis, a single subepidermal cell in the ovule primordium is
specified as the MMC (Vandendries, 1909; Schneitz et al., 1995).
The MMC enters meiosis and gives rise to four haploid cells, of
which only one survives to develop a into female gametophyte. To
further characterize the timeframe of cell differentiation in the apical
region of the developing ovule, we correlated premeiotic to postmeiotic cellular differentiation with integumentary growth in five
genetically distant ecotypes of Arabidopsis and their respective
hybrids (Figure 1). In addition to the reference ecotype Columbia-0
(Col-0), we selected Shakdara-0 (Sha-0), Borky-4 (Bor-4), Cape
Verde Islands-0 (Cvi-0), and Monterrosso-0 (Mr-0) as four genetically distant ecotypes with a contrasting pattern of geographic and
environmental distribution (Nordborg et al., 2005). As presented in
Figure 1A and Table 1, four temporal stages that encompass
megasporogenesis were defined to score cell differentiation and
division. We defined Stage 1 as corresponding to ovule primordia
having a well-defined proximal-distal axis and the absence of integument initiation. Stage 2 comprises ovules that have initiated
integument growth and have an inner integument composed of
a maximum of two cell layers. In Stage 3 ovules, both integuments
have initiated growth and development, and the inner integument
has three to four cell layers. Finally, Stage 4 ovules contain an inner
integument of at least five cell layers and an adaxial outer integument that reaches the tip of the nucellus.
All four stages encompass the main cellular events occurring
during megasporogenesis, up to the differentiation of the FM at
the onset of megagametogenesis. For Stages 1 to 3, the majority of
ovules in all five ecotypes showed a single conspicuous MMC with
a dense cytoplasm, large nucleus, and prominent nucleolus, occupying a preponderant position in direct contact with the apical L1
layer. At these same stages, all five ecotypes also showed variable
frequencies of ovules harboring more than one cell reminiscent of
the MMC (Figures 1B to 1E); these alternative morphological events
were named ectopic configurations. Mr-0 was the most variable
ecotype, with nearly 30% of ovules showing ectopic configurations
at Stage 1 (Table 1; Supplemental Table 1). Following the developmental progression from Stages 1 to 4, the frequency of ectopic configurations declined in all ecotypes, being almost absent
at Stage 4, suggesting developmental competition between neighboring ectopic cells (Table 1). In all cases where ectopic configurations were observed, a single linear arrangement of degenerated
cells adjacent to a surviving megaspore was observed at Stage 4,
suggesting that a single cell divided meiotically.
We also quantified cell differentiation during megasporogenesis
in F1 individuals resulting from intraspecific crosses between Col-0
and each of the additional four selected ecotypes. The results were
analyzed using a x2 test. As shown in Table 1 and Supplemental
Table 2, the phenotypic frequencies revealed cases of both additive
and nonadditive effects, depending on the parental combinations
and the developmental stage analyzed. When compared with their
mid-parent value (i.e., the average phenotype frequency observed in
the parental lines), F1 individuals resulting from crosses between
Col-0 and Sha-0 showed additive effects in Stages 1, 3, and
4, while F1 plants from crosses between Col-0 and Bor-4 or Cvi-0
ecotypes exhibited nonadditive phenotypic frequencies at different
stages. Whereas for Col-0 3 Cvi-0 F1 individuals nonadditivity was
confirmed for Stages 1 and 3, Col-0 3 Bor-4 F1 hybrids showed the
same pattern from Stages 1 to 3. A statistically significant suppressive effect on the frequency of ectopic configurations was
observed in the Col-0 3 Sha-0 F1 individuals, suggesting that in this
cross nonadditive effects are associated with reduction of ectopic
configurations. In the case of Col-0 3 Mr-0 F1 hybrids, a nonadditive effect was only present across Stages 1 and 2. In all genetic
combinations, the trend in phenotypic frequencies was consistent
in reciprocal crosses, showing that the selection of an ecotype as a
maternal or paternal parent did not influence the F1 results
(Supplemental Table 1).
Finally, we quantified cell differentiation during megasporogenesis in tetraploid individuals of Col-0 and Landsberg erecta (Ler).
Both ecotypes exhibited high frequencies of ovules showing
Epigenetic Variation and Gametogenesis
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Figure 1. Ovule Morphology and Gamete Precursor Cell Differentiation at Four Stages of Ovule Development in Arabidopsis.
(A) Temporal stages encompassing megasporogenesis as defined on the basis of integument growth. Asterisk indicates the presence of a degenerated
megaspore. L1, L1 cell layer.
(B) Bor-4 ovule with three differentiated female gamete precursors (arrowheads).
(C) Cvi-0 ovule with two differentiated female gamete precursors (arrowheads).
(D) and (E) Confocal sections showing Mr-0 ovules containing female gamete precursors showing higher intensity of propidium iodide staining
compared with adjacent sporophytic cells.
Bars = 10 mm in (A), 5 mm in inset in (A), and 10 mm in (B) to (E).
ectopic configurations compared with their diploid counterparts
(29.9% for Col-0; 24.2% for Ler at Stage 1). Although in both
ecotypes the frequency of ectopic configurations decreased during
the progression of megasporogenesis, 10.7% of Col-0 ovules
showed ectopic configurations at Stage 4—a frequency significantly higher than diploid ovules of this same ecotype (0.8%) at that
stage—suggesting that dosage factors are able mitigate the developmental mechanisms that favor competition between neighboring gamete precursors. Overall, these results indicate that
premeiotic gamete cell specification is naturally variable in the ovule
of Arabidopsis, with a strong tendency toward restricting the persistence of ectopic configurations at late stages of megasporogenesis. They also show that specific allelic combinations have
a tendency to increase the differentiation of ectopic cells at the
onset of meiosis, whereas higher ploidy levels in specific ecotypes
tend to increase the prevalence of ectopic cells at late stages of
megasporogenesis.
The Frequency of Ectopic Cells in the Ovule Segregates
Continuously among Individuals of Wild-Type
F2 Populations
To determine whether multiple segregating factors could contribute
to the variability found in gamete precursor cell specification, the
frequency of ovules showing ectopic configurations was quantified
in a population of F2 individuals from two different crosses that
previously exhibited nonadditive effects (Col-0 3 Cvi-0 and Col-0 3
Bor-4). For ;50 F2 individuals per cross, close to 100 ovules were
cytologically analyzed at Stage 1 in both segregating populations
(Figure 2). In both cases, the phenotypic frequencies of ectopic
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Table 1. Quantitative Analysis of Cell Differentiation during Megasporogenesis in Ovules of Arabidopsis Ecotypes, Their Wild-Type Hybrid, and
Tetraploid Lines
Stage 1
Genotype
Col-0
Ler
Sha-0
Bor-4
Cvi-0
Mr-0
Col-0 3
Col-0 3
Col-0 3
Col-0 3
Col 4N
Ler 4N
Stage 2
Ectopic
Configurations
Single
Single MMC (%) (%)
Total MMC (%)
478
282
379
620
314
303
Sha-0 F1 491
Bor-4 F1 323
Cvi-0 F1 174
Mr-0 F1
401
282
292
(90.4)
(83.7)
(95.2)
(89.5)
(88.5)
(72.7)
(88.2)
(78.6)
(71.3)
(77.4)
(70.1)
(75.8)
51 (9.6)
55 (16.3)
19 (4.8)
73 (10.5)
41 (11.5)
114 (27.3)
66 (11.8)
88 (21.4)
70 (28.7)
117 (22.6)
120 (29.9)
93 (24.2)
529
337
398
693
355
417
557
411
244
518
402
385
578
325
705
702
335
339
612
311
305
251
534
335
(89.6)
(89.5)
(96.6)
(93)
(92.5)
(82.9)
(92)
(79.7)
(86.9)
(83.9)
(85.9)
(82.7)
Stage 3
Stage 4
Ectopic
Configurations
Single
(%)
Total MMC (%)
Ectopic
Ectopic
Configurations
Functional
Configurations
(%)
Total Megaspore (%) (%)
Total
67
38
25
53
27
70
13
79
46
48
88
70
24 (3.9)
10 (3.92)
4 (0.8)
16 (4.6)
16 (6.2)
13 (3.7)
12 (2.6)
26 (8.8)
28 (10.1)
21 (6.3)
10 (2.7)
6 (2.3)
(10.4)
(10.5)
(3.4)
(7)
(7.5)
(17.1)
(8)
(20.3)
(13.1)
(16.1)
(14.1)
(17.3)
configurations followed a normal distribution, broadly coalescing in
seven distinct phenotypic classes (Shapiro-Wilk test for normality,
P > 0.7; Figure 2; Supplemental Table 3), with transgression beyond
the maximum limit reached by the parents in both F2 populations
(up to 40%). Although subtle differences in phenotypic frequencies
were observed between the two segregating populations, the
broad categories were conserved, indicating that the same number
of factors in both populations are contributing to the control of the
cell specification trait. This result suggests that several discrete
genetic factors are influencing the specification of gamete precursors during early ovule development in Arabidopsis and that they
contribute additively to phenotype frequency of ovules with ectopic
cells, favoring a quantitative genetic control of cell specification.
Ecotypic Variation in Cell Differentiation Is Influenced by the
Activity of AGO9
The presence of ectopic configurations at variable frequencies in
ecotypes of Arabidopsis is reminiscent of phenotypes found in
dominant ago9 and rdr6 loss-of-function mutants. Plants defective
in AGO9 or RDR6 showed ectopic differentiation of female gamete
precursors and subsequent formation of extranumerary female
529
337
398
693
355
409
625
390
351
299
622
405
593
245
500
335
241
339
453
269
249
313
355
253
(96.1)
(96.1)
(99.2)
(95.4)
(93.8)
(96.3)
(97.4)
(91.2)
(89.9)
(93.7)
(97.3)
(97.7)
529
337
398
693
355
352
465
295
277
334
365
259
257
210
197
287
266
296
190
291
255
174
158
275
(99.2)
(95.5)
(100)
(99)
(100)
(98)
(98.4)
(98.3)
(98.1)
(96.1)
(89.3)
(96.2)
2 (0.8)
10 (4.55)
0 (0)
3 (1)
0 (0)
6 (2)
3 (1.6)
5 (1.7)
5 (1.9)
7 (3.9)
19 (10.7)
11 (3.8)
529
337
398
693
355
302
193
296
260
181
177
286
gametophytes in the developing ovule (Olmedo-Monfil et al., 2010).
To investigate a possible involvement of these sRNA-related genes
in the natural ecotypic variation during megasporogenesis, we
quantified the frequency of ectopic configurations in F1 progeny
resulting from the cross of ago9-3 (Col-0) individuals with Bor-4,
Sha-0, Cvi-0, and Mr-0 plants (Table 2). Whereas heterozygous F1
individuals resulting from a cross between homozygous ago9-3
and Sha-0 showed an additive increase in ovules exhibiting ectopic
configurations (Table 2), F1 plants from homozygous ago9-3
crossed to Bor-4, Cvi-0, and Mr-0 showed no increase in the frequency of ovules showing ectopic configurations compared with
heterozygous ago9-3/+ or their corresponding wild-type ecotypic
hybrids. A similar tendency was detected in reciprocal crosses
between rdr6-15 mutants (Col-0) and Cvi-0, indicating that the effect of dominant ago9 or rdr6 mutations in the ectopic configuration
phenotype is buffered in ecotypic hybrids, as the introduction of
a dominant ago9 or rdr6 allele did not result in an increase in the
number of ovules showing ectopic configurations. These results
confirm that a high frequency of ectopic configurations in F1 hybrids
resulting from crosses between Col-0 and Bor-4 or Cvi-0 is likely
related to divergent allelic combinations. Interestingly, homozygous
ago9-3 F1 hybrids resulting from a cross between ago9-3 and BC4
Figure 2. Segregation of Ovules Showing Ectopic Configurations in F2 Populations Originating from Ecotypic F1 Hybrids.
(A) Frequency of ovules showing ectopic configurations in Col-0 3 Cvi-0 F2 individuals.
(B) Frequency of ovules showing ectopic configurations in Col-0 3 Bor-4 F2 individuals.
Epigenetic Variation and Gametogenesis
heterozygous ago9-3/+ plants introgressed into a Bor-4 background
showed frequencies of ectopic configurations in Stage 1 ovules
equivalent to those found in homozygous ago9-3 (Col-0) individuals
(x2 = 2.35 < x2 0.05[1] = 3.84; Table 2). Since homozygous ago9-3
mutants were previously shown to lack any AGO9 activity (OlmedoMonfil et al., 2010), this result indicates that the buffering effect
described above is overcome by the complete loss of function of
AGO9. Our results suggest that although hybridization between
some phylogenetically distant ecotypes perturbs the process of
gamete precursor cell specification at quantitative levels similar to
those observed in mutants defective in AGO9 function, the introduction of mutations affecting AGO9 or RDR6 activity is buffered
by complex allelic interactions in specific ecotypic F1 hybrids. They
also show that the complete absence of AGO9 activity disrupts this
buffering effect, revealing a genetic interaction between the natural
mechanisms that control ecotypic variation during megasporogenesis and the function of AGO9.
Natural Variation in Genomic Regulatory Regions of AGO9
Results in Changes in Transcriptional Regulation and
Protein Localization
To determine whether differences in AGO9 expression could help
explain differences in the frequency of ectopic configurations found
between Col-0 and Mr-0 ecotypes, we conducted mRNA wholemount in situ hybridization in developing ovules of Col-0 and
Mr-0 individuals using a 149-bp antisense RNA probe corresponding to a Col-0 sequence located in the 39 untranslated region
(Supplemental Figure 1). As expected from previous results, in both
genetic backgrounds, AGO9 mRNA was localized in all cells of the
ovule primordium throughout megasporogenesis. However, under
identical experimental conditions, the level of mRNA expression
was consistently higher in developing ovules of Col-0 compared
with Mr-0 ovules. Because the level of genomic polymorphisms
between Col-0 and Mr-0 within the sequence corresponding to the
selected probe is not sufficient to cause deficiencies in the formation of antisense RNA:mRNA duplexes to explain this difference
(Supplemental Figure 1), these in situ hybridization results suggest
that AGO9 mRNA is weakly expressed in Mr-0 ovules. To determine
whether this difference in AGO9 expression is related to ecotypic
differences in AGO9 transcriptional regulation, the complete 2619-bp
intergenic region upstream of the AGO9 coding sequence isolated
from either Col-0 or Mr-0 plants was cloned in front of the uidA
(b-glucuronidase [GUS]) reporter gene to subsequently transform
Col-0 wild-type individuals. Stage 1 ovules of Col-0 plants transformed with a transcriptional fusion that includes the Col-0 AGO9
regulatory sequence (proCol-0AGO9:GUS) showed strong GUS
expression after 6 h of histochemical incubation, with initial expression in the nucellar cells located at the proximal pole of the
MMC (99%, n = 110). At Stage 2, GUS expression was localized in
additional nucellar cells and in the L1 layer. At Stage 4, GUS expression prevailed in the chalazal region of the ovule (Figures 3A to
3C). In contrast, ovules of Col-0 plants transformed with a transcriptional fusion that includes the Mr-0 AGO9 regulatory sequence
(proMr-0AGO9:GUS) showed GUS expression only after 24 to 36 h
of histochemical incubation in a pattern antagonistic to developing
ovules of proCol-0AGO9:GUS transformants. The majority of
proMr-0AGO9:GUS ovules at Stage 1 (88%, n = 568) showed GUS
expression restricted to L1 cells located at the apical (distal) pole of
the MMC. Although Stage 2 ovules showed GUS expression restricted to a larger number of L1 cells in the same region, Stage 4
ovules showed GUS expression in a pattern similar but not identical
to Stage 4 ovules in proCol-0AGO9:GUS transformants (Figures 3D
to 3F), since GUS expression in the subepidermal apical region
remains strong in proMr-0AGO9:GUS transformants.
We also determined the pattern of AGO9 transcriptional regulation
in developing ovules of F1 plants generated by crossing individuals
of Sha-0, Bor-4, and Cvi-0 to the proCol-0AGO9:GUS Col-0 line
(Figures 3G to 3L). Whereas the general pattern of GUS expression
was reminiscent of the pattern found in ovules of Col-0, ovules of F1
individuals exhibited specific differences at both premeiotic and
postmeiotic stages (Stages 1 and 4). At Stage 1, whereas Sha-0 3
proCol-0AGO9:GUS F1 ovules showed a pattern equivalent to Col-0
(Figure 3G), GUS expression was substantially reduced in Bor-4 3
proCol-0AGO9:GUS and Cvi-0 3 proCol-0AGO9:GUS F1 ovules
(Figures 3H and 3I). Also, whereas initial GUS expression in Col-0
ovules was in most nucellar cells located at the proximal pole of the
MMC, Bor-4 3 proCol-0AGO9:GUS F1 individuals showed initial
expression in a small cluster comprising L2 and L3 cells at the
midlateral region of the premeiotic primordium (Figure 3H). At Stage
4, Bor-4 3 proCol-0AGO9:GUS and Cvi-0 3 proCol-0AGO9:GUS
F1s also showed reduced GUS expression compared with Sha-0 3
Table 2. Quantitative Comparison among Wild-Type and Mutant Ecotypic Hybrids at Stage 1
Genotype
Single MMC (%)
Ectopic Configurations (%)
Total
Col-0 3 Sha-0 F1
ago9-3 3 Sha-0 F1
Col-0 3 Cvi-0 F1
ago9-3 3 Cvi-0 F1
rdr6-15 3 Cvi-0 F1
Col-0 3 Mr-0 F1
ago9-3 3 Mr-0 F1
Col 3 Bor-4 F1
ago9-3 3 Bor-4 F1
ago9-3 3 BC ago9-3 (Bor-4) 2/2 F1
ago9-3/+
rdr6-15/+
ago9-3
491
646
174
228
372
401
303
323
560
396
371
369
551
66 (11.8)
178 (21.6)
70 (28.7)
72 (24.0)
111 (23)
117 (22.6)
111 (26.8)
88 (21.4)
154 (21.6)
140 (26.1)
122 (24.7)
169 (31.4)
236 (30)
557
824
244
300
483
518
414
411
714
536
493
538
787
(88.2)
(78.4)
(71.3)
(76.0)
(77)
(77.4)
(73.2)
(78.6)
(78.4)
(73.9)
(75.3)
(68.6)
(70)
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Figure 3. Reporter Gene Expression in proCol-0AGO9:GUS and proMr-0AGO9:GUS Transgenic Lines in a Col-0 Background.
(A) Stage 1 ovule of a transgenic proCol-0AGO9:GUS plant.
(B) Stage 2 ovule of a transgenic proCol-0AGO9:GUS plant.
(C) Stage 4 ovule of a transgenic proCol-0AGO9:GUS plant.
(D) Stage 1 ovule of a transgenic proMr-0AGO9:GUS plant.
(E) Stage 2 ovule of a transgenic proMr-0AGO9:GUS plant.
(F) Stage 4 ovule of a transgenic proMr-0AGO9:GUS plant.
(G) Stage 1 ovule of a Sha-0 3 proCol-0AGO9:GUS (Col-0) F1 plant.
(H) Stage 1 ovule of a Bor-4 3 proCol-0AGO9:GUS (Col-0) F1 plant.
(I) Stage 1 ovule of a Cvi-0 3 proCol-0AGO9:GUS (Col-0) F1 plant.
(J) Stage 4 ovule of a Sha-0 3 proCol-0AGO9:GUS (Col-0) F1 plant.
(K) Stage 4 ovule of a Bor-4 3 proCol-0AGO9:GUS (Col-0) F1 plant.
(L) Stage 4 ovule of a Cvi-0 3 proCol-0AGO9:GUS (Col-0) F1 plant.
The nucellar region is highlighted by dashed lines in (A). Arrows indicate the presence of gamete precursors in (A), (B), (D), (E), and (G) to (I), and the
functional megaspore is highlighted in (C) and (F). Asterisks indicate the presence of degenerated megaspores in (C) and (F). Bars = 10 mm in (A), (B),
(D), (E), and (G) to (I) and 20 mm in (C), (F), and (J) to (L).
proCol-0AGO9:GUS F1 and Col-0 ovules (Figures 3J to 3L), indicating that in these F1 individuals AGO9 expression is reduced
throughout megasporogenesis. Taken together, these results suggest that the factors controlling transcriptional regulation of AGO9
are variable among ecotypes and their hybrids. They also suggest
that although divergent regulation of the Mr-0 AGO9 promoter region
in the Col-0 background can cause antagonistic changes in the
spatial pattern of reporter gene expression, additional genetic factors
are likely to compensate these changes to produce similar but not
identical spatial patterns of transcriptional regulation at subsequent
stages of ovule development.
To determine the pattern of AGO9 protein expression in ovules of
selected ecotypes, we conducted whole-mount immunolocalizations using a polyclonal antibody previously reported to specifically
recognize an epitope of the AGO9 protein. Previous experiments
showed that in wild-type Col-0 ovules undergoing meiosis, AGO9
is localized in discrete cytoplasmic foci of sporophytic cells, preferentially in the apical region of the L1 layer, but not in meiotically
dividing cells or in the functional megaspore. The same antibody
was used to localize AGO9 in Stage 1 ovules of all five selected
ecotypes (Figure 4). In premeiotic Stage 1 ovules of Col-0, Bor-4,
and Sha-0, AGO9 was localized in cytoplasmic foci present in most
sporophytic cells of the primordium, but also transiently in the
nucleus of the MMC at variable expression levels. In these three
ecotypes, most Stage 1 ovules also showed a clear pattern of
preferential AGO9 localization in a cluster of apical L1 cells located
at the distal pole of the MMC, in agreement with previous immunolocalizations conducted in Col-0 at subsequent developmental
stages (Figures 4A to 4C). By contrast, most Stage 1 ovules of Mr-0
and Cvi-0 did not show this preferential localization of AGO9 in cells
of the L1 layer (Figures 4D and 4E). Whereas the large majority of
ovules of Col-0, Bor-4, and Sha-0 showed AGO9 localization in
apical cells of the L1 layer (86.4% for Col-0, 97.2% for Bor-4, and
95.8% for Sha-0; total n = 119), only close to 50% showed the
equivalent pattern in Mr-0 and Cvi-0 (50% for Cvi-0 and 53.4% for
Mr-0; total n = 94). These results indicate that the ecotypic differences found in the pattern of AGO9 transcriptional regulation are
also reflected in the cellular pattern of protein localization.
Abnormal Ectopic Cells in Ovules Defective for RDR6 Share
Identity with Ectopic Cells Found in the Analyzed Ecotypes
The nuclear pattern of AGO9 protein localization specifically shown
by the MMC was also present in extranumerary cells found in
ovules of ecotypes Mr-0, Bor-4, and Cvi-0. Previous studies
indicated that heterozygous rdr6-15/+ individuals showed aberrant
Epigenetic Variation and Gametogenesis
cell specification at premeiotic stages, with several female gamete
precursors differentiating, growing, and dividing at the apical region
of the developing ovule. To determine if the ectopic cells naturally
found in selected ecotypes acquire a developmental identity similar
to aberrant accessory cells found in ovules defective for RDR6, we
conducted AGO9 whole-mount immunolocalizations in ovules of
heterozygous rdr6-15/+ plants. Under whole-mount histological
observations, the frequency at which Stage 1 rdr6-15/+ ovules
exhibited accessory cells was of 31% (n = 538). For rdr6-15/+
ovules, whereas the preferential localization of AGO9 in the apical
cells of the L1 layer was absent, AGO9 was expressed in the nucleus of accessory cells present in the nucellus (Figure 4F). Under
confocal illumination, the frequency of Stage 1 rdr6-15/+ ovules
showing AGO9 localization was of 26% (n = 33). An identical nuclear pattern of AGO9 localization was observed in Stage 1 ectopic
cell configurations present in ovules of the Mr-0, Bor-4, and Cvi-0
ecotypes (Figures 4G to 4I), suggesting that naturally occurring
ectopic cells in Arabidopsis ecotypes acquire the same identity as
abnormal female gamete precursors found in rdr6-15/+ individuals,
confirming that natural variation in the specification of female
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gamete precursors acts through some of the sRNA-dependent
epigenetic pathways that prevail in the ovule of Arabidopsis.
DISCUSSION
Intraspecific natural variation in the angiosperms offers opportunities for natural selection to exert evolutionary pressure over the
diversity of developmental pathways that are essential for survival,
reproduction, or dispersal (Alonso-Blanco et al., 2009; Prasad et al.,
2012; Anderson et al., 2014). In some cases, this variation is directly
related to quantitative traits that can be mapped and associated
with developmental or physiological processes (Atwell et al., 2010;
Strange et al., 2011), whereas in others it can be traced to functional multiallelic diversity of a single locus (Todesco et al., 2010).
Epigenetic natural variation, manifested through either epimutations
or epialleles, is currently under intense investigation to assess its
impact in both adaptability and evolution. Although the extent to
which epigenetic variation contributes to phenotypic variation remains to be determined, several examples of naturally occurring
epialleles that affect plant development have been reported (Brink,
Figure 4. AGO9 Protein Localization in Stage1 Ovules of Arabidopsis.
(A) AGO9 localization in a Col-0 ovule.
(B) AGO9 localization in a Sha-0 ovule.
(C) AGO9 localization in a Bor-4 ovule.
(D) Cvi-0 ovule showing absence of AGO9 localization in the L1 layer.
(E) Mr-0 ovule showing absence of AGO9 localization in the L1 layer.
(F) AGO9 localization in a heterozygous rdr6-15/+ ovule; arrows indicate the presence of gamete precursors.
(G) AGO9 localization in a Mr-0 ovule; arrows indicate the presence of gamete precursors.
(H) AGO9 localization in a Bor-4 ovule; arrows indicate the presence of gamete precursors.
(I) AGO9 localization in a Cvi-0 ovule; arrows indicate the presence of gamete precursors.
(J) AGO9 expression is absent in homozygous ago9-3 ovules.
L1, L1 cell layer; N, nucleus of the MMC. Bars = 10 mm in (A) to (J) and 5 mm in insets in (F) to (I).
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1956; Cubas et al., 1999; Manning et al. 2006). Arabidopsis has
provided abundant evidence of epigenetic variation through either
spontaneous or induced epimutants, including the induction of
flowering, seed development, and genomic imprinting (Shindo
et al., 2006; Fujimoto et al., 2011; Pignatta et al., 2014); however,
the consequences of natural epigenetic variation in gametogenesis
have not been investigated.
Our study shows that natural variation among distinct Arabidopsis ecotypes reflects the epigenetic mechanisms that lead to
the differentiation of female gamete precursors at the onset of
meiosis. Despite significant differences in their intrinsic frequencies
of ectopic configurations, crosses between Col-0 and Sha-0 or
Mr-0 showed additive effects, suggesting that widespread natural
variation is based on conserved and independent genetic factors
that control the specification of gamete precursors. By contrast, F1
hybrids between Col-0 and Bor-4 and Cvi-0 gave rise to ovules in
which the frequency of gamete precursors was significantly increased compared with the corresponding mid-parent value. This
disruption in the control of cell specification, beyond the frequency
observed in the parental ecotypes, is reminiscent of nonadditive
effects associated with the phenomenon of heterosis or hybrid
vigor (Birchler et al., 2010; Chen, 2010). Differences in the phenotypic frequency of ectopic configurations are reminiscent of heterotic responses previously reported for F1 progeny of some
Arabidopsis ecotypes (Moore and Lukens, 2011; Groszmann et al.,
2014). A similar effect was also found in Medicago sativa, for which
only certain parental combinations produced heterotic responses,
as revealed by nonadditive gene expression (Li et al., 2009). Although an increase in the number of female gamete precursors
does not necessarily affect fertility or seed production, the possibility of increasing the number of cells entering the gametophytic
developmental pathway, through divergent allelic combinations
present in specific ecotypic hybrids, could represent an adaptation
to detrimental conditions that affect female meiosis and cause
fertility defects.
Several studies have raised the possibility that alternative reproductive pathways, such as tetraspory or asexual reproduction
through seeds (apomixis), evolved as a response to hybridization,
genomic collisions, or unstable climatic environment (Carman,
1997; Voigt-Zielinski et al., 2012; Lovell et al., 2013; Hojsgaard
et al., 2014). Segregating F2 populations of ecotypic crosses
showed a continuous distribution of the frequency of ectopic
configurations, suggesting that the genetic factors influencing the
phenotype are quantitative and conserved among different ecotypes. In agreement with our results, these type of nonadditive
effects are particularly evident in ecotypic hybrids or polyploid individuals that are often correlated with changes in gene expression
(Chen, 2007; Miller et al., 2012; Chen, 2013), and large-scale
transcriptional analysis in hybrids of Arabidopsis ecotypes indicate
that nonadditive changes in gene expression result in alteration of
developmental or physiological processes, including photosynthetic
capacity, seedling development, or leaf morphology (Fujimoto et al.,
2012; Meyer et al., 2012; Todesco et al., 2012; Chen, 2013).
In addition to genetically related natural variation, epigenetic
variability can also contribute to the phenotypic differences found
among Arabidopsis ecotypes (Latzel et al., 2013; Silveira et al.,
2013; Cortijo et al., 2014). Our results show that ecotypic variation
in cell differentiation is influenced by the functional activity of AGO9
and RDR6. Whereas AGO9 encodes an AGO protein that preferentially interacts with a 24-nucleotide sRNA corresponding to
transposable elements located in the pericentromeric regions of all
Arabidopsis chromosomes (Durán-Figueroa and Vielle-Calzada,
2010; Olmedo-Monfil et al., 2010), RDR6 acts in the biogenesis of
various types of sRNAs including trans-acting small interfering
RNAs and in posttranscriptional transgene silencing mediated by
sRNAs (Peragine et al., 2004; Yoshikawa et al., 2005; Chitwood
et al., 2009). AGO9 is phylogenetically related to AGO4 and AGO6,
proteins that are involved in heterochromatic silencing through
the RNA-directed DNA methylation pathway (Zilberman et al.,
2003, 2004; Vaucheret, 2008; Havecker et al., 2010; Mallory and
Vaucheret, 2010; Eun et al., 2011). The introduction of a single
dominant loss-of-function ago9 or rdr6 allele in some Arabidopsis
ecotypes resulted in a buffering effect that limited the number of
ovules showing ectopic configurations, whereas this buffering
effect was lost in plants that completely lost AGO9 activity.
These results suggest that the partial loss of AGO9 activity is
epigenetically sensed to restrict the number of sporophytic cells
that premeiotically differentiate into female gamete precursors
by AGO9- or RDR6-dependent mechanisms yet to be elucidated.
This is also reflected in the comparison of the pattern of reporter
gene expression that results from GUS fusions to a large 2619-bp
genomic segment that represents the AGO9 regulatory region of
either Col-0 or Mr-0. Variation in transcriptional regulation of AGO9
was observed when the AGO9 regulatory region present in the
genome of Mr-0 was introduced into the Col-0 background, exposing the effects of accumulated genetic differences between
these two ecotypes. Although the pattern of AGO9 mRNA localization is equivalent in both ecotypes, their initial pattern of transcriptional regulation is antagonistic, revealing that divergent allelic
combinations can have an effect at the transcriptional level in the
control of AGO9 expression. When comparing transcriptional fusions corresponding to Col-0 and Mr-0 alleles, the pattern of GUS
expression is similar but not identical at the onset of female gametogenesis, suggesting that a dynamic pattern of transcriptional
regulation and possibly mRNA transport prevails during megasporogenesis, since the pattern of mRNA localization coincides
with the final pattern of AGO9 protein localization. Because AGO9
contains a large intron at the 59 region that was not included in the
proAGO9:GUS fusions tested, it is possible that additional genomic
elements absent in our transgenic lines could influence the ecotypic pattern of AGO9 transcription. The transcriptional regulation
of AGO9 is also altered in some hybrids between ecotypes, particularly in those exhibiting a nonadditive exacerbation of the frequency of ectopic configurations (Col-0 3 Bor-4 and Col-0 3 Cvi-0
F1 individuals), suggesting that hybridization can also perturb
AGO9 expression by reducing its transcriptional activity, leading to
the differentiation of accessory cells into premeiotic gamete precursors, leading to phenotypic consequences that result in natural
variation during female reproductive development.
Previous results showed that during megasporogenesis,
AGO9 protein localization was confined to discrete foci present
in the cytoplasm of sporophytic cells within the nucellus, with
abundant expression at the apex of the ovule primordium, in cells
of the L1 layer (Olmedo-Monfil et al., 2010). Our results show that
the same pattern prevails in Stage 1 ovules at the onset of meiosis;
however, the nucleus of the MMC in several ecotypes sporadically
Epigenetic Variation and Gametogenesis
shows AGO9 expression, suggesting that an ephemeral and transient nuclear AGO9 localization can be found in the MMC, a discovery that suggest a dynamic pattern of protein traffic between
cytoplasm and nucleus reminiscent of AGO4 dynamics in root cells
(Li et al., 2006; Pontes et al., 2006; Ye et al., 2012). In the case of
AGO9, nuclear localization is exclusive of the MMC and ectopic
cells acquiring a gamete precursor identity in several ecotypes and
individuals defective in RDR6, a result suggesting that ectopic cell
configurations naturally exhibited by some Arabidopsis ecotypes
share a developmental identity with ectopic gamete precursors
found in ovules defective in the epigenetic control of cell specification. Further cytological and molecular studies will be necessary
to integrate an understanding of AGO9 function, sRNA dynamics,
and the early control of cell specification in the ovule.
Contrary to other cases in which specific ecotype combinations
resulted in hybrids showing deleterious phenotypes (Todesco et al.,
2010; Durand et al., 2012), no detrimental effects in fertility were
found in ecotypic hybrids or AGO9-defective plants, suggesting
that the phenotypic variation among ecotypes could be related to
neutral accumulation of genetic differences or by episodes of
natural selection that lead to fixation of certain genetic configurations (Atwell et al., 2010; Long et al., 2013). While several ecotypes and their hybrids exhibited variable frequencies of ectopic
configurations, female gametophyte development occurred normally in all backgrounds, with a single meiotically derived cell differentiating into a functional megaspore, suggesting that in most
cases ectopic cells degenerate without additional development. A
similar pattern was observed in Sorghum bicolor, where the sporadic presence of aposporous initials correlated with an acceleration of meiotic initiation but no extranumerary female gametophytes
were found at subsequent developmental stages (Carman et al.,
2011). Our results favor the possibility of a flexible control of
gamete precursor cell specification prevailing in Arabidopsis at the
onset of meiosis, but a robust canalization mechanism acting at the
onset of the processes that trigger meiosis and megagametogenesis; the molecular mechanisms that ensure the establishment
of such a canalized developmental process could be partially dependent on the epigenetic pathways that involve AGO9 and RDR6.
In several families of flowering plants, the number of female gamete
precursors that can occur within a single ovule is highly variable,
with extreme examples such as Peonia californica exhibiting up to
40 premeiotic precursors (Walters, 1962). Our results suggest that
some of the epigenetic mechanisms that prevail during early ovule
development are involved in the natural phenotypic variation observed during megasporogenesis. Future comparative studies of
the role of genes such as AGO9 and RDR6 in species showing
alternative gametophytic pathways will provide insights into the
evolution of the epigenetic mechanisms that control female gametogenesis and cell specification in flowering plants.
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were also obtained from the ABRC. Five rounds of backcrossing were
performed to introgress the ago9-3 mutant allele into Bor-4 ecotype. All seeds
were surface sterilized with 100% ethanol or with chlorine gas and germinated
in Murashige and Skoog medium at 22°C or 25°C under stable long-day (16 h
light/8 h dark) or full-day conditions. Plants were grown at 24°C under
controlled growth chamber or greenhouse conditions.
Generation and Analysis of proAGO9:GUS Transformants
The regulatory region of the AGO9 gene (At5g21150) from Col-0 and Mr-0
ecotypes was transcriptionally fused to the uidA (GUS) reporter gene by
amplifying a 2619-bp DNA fragment corresponding to the complete intergenic
region located upstream of its coding sequence but excluding the 59 untranslated region (primers: pAGO9S1_HindIII 59-AATATTAAGCTTGGGAGACAGAAAGTGCGGTGAGAGAGAGAC-39 and pAGO9AS1_KpnI
59-GGCCTGGGTACCATTCACTAAAATATAGGTGTGTCGCTTATA-39). Amplicons were cloned into pCR8 TOPO TA (Invitrogen) and used as donors in
LR recombination (LR Clonase II; Invitrogen) with pMDC162 (Curtis and
Grossniklaus, 2003), producing a binary vector that contains the uidA reporter
gene. Transgenic Col-0 plants were obtained by floral dipping as previously
described (Clough and Bent, 1998). At least 15 T1 individuals were obtained
and analyzed for each of the two constructs (Col-0 or Mr-0 version). At least
five individuals of 10 independent T2 transformants were cytologically examined to quantify the frequency of GUS expression at different stages of
megasporogenesis.
Cytological and Histochemical Analysis
Inflorescences were fixed in FAA (50% ethanol, 10% formaldehyde, and
5% acetic acid) for 24 to 48 h and subsequently dehydrated in 70%
ethanol. Immature flower buds were dissected with hypodermic needles
(1-mL insulin syringes; TERUMO SS10M2913M) to isolate the developing
gynoecia. Several hundred ovules for each of the four stages were obtained
by mounting individual gynoecia in regular microscope slides and immersing
in Herr’s clearing solution (phenol:chloral hydrate:85% lactic acid:xylene:clove
oil in a 1:1:1:0.5:1 proportion). Histochemical localization of GUS activity was
performed by incubating gynoecia of 0.5 to 1 mm in length in GUS staining
solution (10 mM EDTA, 0.1% Triton X-100, 5 mM potassium ferrocyanide,
5 mM potassium ferricyanide, and 1 mg mL21 5-bromo-4-chloro-3-indolylb-D-glucuronic acid in 50 mM sodium phosphate buffer, pH 7.4) for 2 to 36 h at
37°C. For propidium iodide staining, gynoecia of 0.5 to 0.7 mm in length were
fixed in FPA (10% formaline, 5% propionic acid, and 70% ethanol) overnight
at 4°C. After fixation, samples were washed with 100 mM L-arginine, pH 8.0
(Sigma-Aldrich) and stained with 2 mg mL21 propidium iodide in 100 mM of
L-Arg (pH 12). Ovule primordia were exposed by gently pressing a cover slip
over a conventional slide containing 16 mL of Vectashield (VectorLabs). Serial
optical sections were obtained on a Zeiss LSM510 META confocal laser
scanning microscope, with single-track configuration for detecting propidium
iodide (excitation with a diode-pumped solid-state laser at 568 nm, with
emission collected using a band-pass of 575 to 615 nm). Sections were edited
using ImageJ software (Schneider et al., 2012). For light microscopy observations, samples were analyzed under Nomarski illumination using a DMR
Leica microscope.
Whole-Mount in Situ Hybridization
METHODS
Plant Material and Growth Conditions
Arabidopsis thaliana ecotypes Bor-4, Sha-0, Cvi-0, and Mr-0 were obtained from the ABRC as part of the collection of 96 ecotypes provided by
Joy Bergelson, Martin Kreitman, and Magnus Nordborg (Stock CS22660).
Tetraploid Col-0 (CS3176) and Ler were a gift from David Galbraith
(University of Arizona). ago9-3 (SAIL_34_G10) and rdr6-15 (SAIL_617_H07)
Digoxigenin-labeled RNA probes specific for AGO9 were synthesized by
in vitro transcription as previously described (Vielle-Calzada et al., 1999;
Olmedo-Monfil et al., 2010). Hybridizations were performed as previously
described (García-Aguilar et al., 2005). Developing gynoecia of 0.5 to 0.8
mm in length were fixed in paraformaldehyde (4% paraformaldehyde, 2%
Triton, and 13 PBS in diethylpyrocarbonate [DEPC]-treated water) for 2 h at
room temperature with gentle agitation, washed three times in 13 PBS-DEPC
water, and embedded in 15% acrylamide:bisacrylamide (29:1) using
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The Plant Cell
precharged slides (Fisher Probe-On) treated with poly-L-Lys as described
(Bass et al., 1997). Gynoecia were gently opened to expose the ovules by
pressing a cover slip on top of the acrylamide. Samples were then treated with
0.2 M HCl for 20 min at room temperature, followed by a washing step in 13
PBS-DEPC water. The slides were then incubated with proteinase K (1 mg
mL21) for 30 min at 37°C. The proteinase K reaction was stopped in glycine
(2 mg mL21). A postfixation step was performed in 4% formaldehyde for
20 min at room temperature, followed by incubation in hybridization buffer (63
SSC buffer, 3% SDS, 50% formamide, and 0.1 mg mL21 of yeast tRNA
[Roche]) for 2 h at 55°C. An overnight incubation was performed using 600 ng
of sense or antisense RNA probe against AGO9 in hybridization buffer. After
overnight incubation, three washing steps with 0.23 SSC/0.1% SDS at 55°C
were performed. Slides were treated with RNase (10 mg mL21), washed four
times in 23 SSC/0.1% SDS at 55°C, and treated with 13 TBS/0.5% blocking
agent (Roche) for 2 h at room temperature. Samples were incubated with antiDIG (Roche) at a concentration of 1:1000 in 13 TBS/1% BSA for 2 h at room
temperature. After antibody incubation, four washing steps in 13 TBS/0.5%
BSA/1% Triton X-100 were performed at room temperature. Slides were
incubated in detection buffer (100 mM Tris, pH 9.5, 100 mM NaCl, 50 mM
MgCl2, 0.1% Tween, and 1 mM levamisole [Sigma-Aldrich]) for 15 min before
adding 10 mL of each nitroblue tetrazolium and 5-bromo-4-chloro-39indolylphosphate (AP Conjugate Substrate Kit; Bio-Rad) per milliliter of detection buffer and incubated overnight at room temperature. Slides were
mounted in 50% glycerol and visualized using Nomarski illumination under
a Leica DRM microscope.
Supplemental Data
Supplemental Figure 1. AGO9 mRNA localization in the developing
ovule of Col-0 and Mr-0 ecotypes.
Supplemental Table 1. Quantitative analysis of ectopic configurations
during megasporogenesis in selected ecotypes of Arabidopsis.
Supplemental Table 2. Quantitative analysis of ectopic configurations
in the ovule of ecotypic F1 hybrids.
Supplemental Table 3. Segregation analysis in F2 populations from
Arabidopsis ecotype hybrids.
ACKNOWLEDGMENTS
We thank Stewart Gillmor and Shai Lawit for useful comments on the
article, Marcelina García-Aguilar for technical advice with immunolocalizations, and members of the Group of Reproductive Development and
Apomixis for stimulating discussions. All Arabidopsis seed stocks were
obtained through the ABRC at Ohio State University. D.R-L. and G.L-M.
were recipients of a graduate scholarship from the Consejo Nacional de
Ciencia y Tecnologia (CONACyT). Research was supported by CONACyT, the DuPont Pioneer regional initiatives to benefit local subsistence
farmers, and the Howard Hughes Medical Institute.
Whole-Mount Protein Immunolocalization
AUTHOR CONTRIBUTIONS
Developing gynoecia of 0.5 to 0.6 mm in length were fixed in paraformaldehyde (13 PBS, 4% paraformaldehyde, and 2% Triton), under
continuous agitation for 2 h on ice, washed three times in 13 PBS, and
embedded in 15% acrylamide:bisacrylamide (29:1) over precharged slides
(Fisher Probe-On) treated with poly-L-Lys as described (Bass et al., 1997).
Gynoecia were gently opened to expose ovules by pressing a cover slip on
top of the acrylamide. Samples were digested in an enzymatic solution
composed of 1% driselase, 0.5% cellulase, and 1% pectolyase (all from
Sigma-Aldrich) in 13 PBS for 60 min at 37°C, subsequently rinsed three
times in 13 PBS, and permeabilized for 2 h in 13 PBS:2% Triton. Blocking
was performed with 1% BSA (Roche) for 1 h at 37°C. Slides were then
incubated overnight at 4°C with AGO9 primary antibody used at a dilution of
1:100 (Olmedo-Monfil et al., 2010). Slides were washed for 8 h in 13
PBS:0.2% Triton, with refreshing of the solution every 2 h. The samples
were then coated overnight at 4°C with secondary antibody Alexa Fluor 488
(Molecular Probes) at a concentration of 1:300. After washing in 13
PBS:0.2% Triton for at least 8 h, the slides were incubated with propidium
iodide (500 mg mL21) in 13 PBS for 20 min, washed for 40 min in 13 PBS,
and mounted in Prolong medium (Molecular Probes) overnight at 4°C. Serial
sections on Stage 1 ovules were captured on a confocal laser scanning
microscope (Zeiss LSM 510 META), with multitrack configuration for detecting iodide (excitation with a diode-pumped solid-state laser at 568 nm,
emission collected using a band-pass of 575 to 615 nm) and Alexa 488
(excitation with an argon laser at 488 nm, emission collected using a bandpass of 500 to 550 nm). Laser intensity and gain were set at similar levels for
all experiments. Projections of selected optical sections were generated
using ImageJ (Schneider et al., 2012).
D.R.-L. and J.-P.V.-C. designed the research. D.R.-L. performed the
genetic crosses and cytological analysis on all materials. D.R.-L. and
G.L.-M. generated the transgenic lines and carried out whole-mount
immunolocalizations and whole-mount in situ hybridizations. U.A.-V.
contributed new histocytochemical tools. D.R.-L. and J.-P.V.-C. analyzed the results and wrote the article.
Accession Numbers
Sequence data for genes in this article can be found in the GenBank/EMBL
database or the Arabidopsis Genome Initiative database under the following
accession numbers: AGO9, NM_122122/AED92940 or At5g21150; and
RDR6, NM_114810/AEE78550 or At3g49500. The ago9-3 (SAIL_34_G10)
and rdr6-15 (SAIL_617_H07) lines were obtained from the ABRC.
Received October 9, 2014; revised January 23, 2015; accepted March 1,
2015; published March 31, 2015.
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Natural Variation in Epigenetic Pathways Affects the Specification of Female Gamete Precursors
in Arabidopsis
Daniel Rodríguez-Leal, Gloria León-Martínez, Ursula Abad-Vivero and Jean-Philippe Vielle-Calzada
Plant Cell; originally published online March 31, 2015;
DOI 10.1105/tpc.114.133009
This information is current as of June 14, 2017
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